Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue.

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

GPER-dependent estrogen signaling increases cardiac GCN5L1 expression.

Reversible lysine acetylation regulates the activity of cardiac metabolic enzymes, including those controlling fuel substrate metabolism. Mitochondrial-targeted GCN5L1 and SIRT3 have been shown to regulate the acetylation status of mitochondrial enzymes, but the role that lysine acetylation plays in driving metabolic differences between male and female hearts is not currently known. In this study, we describe a significant difference in GCN5L1 levels between male and female mouse hearts, and in the hearts of women between post- and premenopausal age. We further find that estrogen drives GCN5L1 expression in a cardiac cell line and uses pharmacological approaches to determine the mechanism to be G protein-coupled estrogen receptor (GPER) activation, via translational regulation.NEW & NOTEWORTHY We demonstrate here for the first time that mitochondrial protein acetylation is increased in female hearts, associated with an increase in GCN5L1 levels through a GPER-dependent mechanism. These findings reveal a new potential mediator of divergent cardiac mitochondrial function between men and women.

Loss of the mitochondrial phosphate carrier SLC25A3 induces remodeling of the cardiac mitochondrial protein acylome.

Mitochondria are recognized as signaling organelles, because under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased posttranslational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel cross talk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction.

RATIONALE: Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. OBJECTIVE: Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. METHODS AND RESULTS: To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). CONCLUSION: The findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

Loss of GCN5L1 in cardiac cells disrupts glucose metabolism and promotes cell death via reduced Akt/mTORC2 signaling.

GCN5L1 regulates protein acetylation and mitochondrial energy metabolism in diverse cell types. In the heart, loss of GCN5L1 sensitizes the myocardium to injury from exposure to nutritional excess and ischemia/reperfusion injury. This phenotype is associated with the reduced acetylation of metabolic enzymes and elevated mitochondrial reactive oxygen species (ROS) generation, although the direct molecular targets of GCN5L1 remain largely unknown. In this study, we sought to determine the mechanism by which GCN5L1 impacts energy substrate utilization and mitochondrial health. We find that hypoxia and reoxygenation (H/R) leads to a reduction in cell viability and Akt phosphorylation in GCN5L1 knockdown AC16 cardiomyocytes, in parallel with elevated glucose utilization and impaired fatty acid use. We demonstrate that glycolysis is uncoupled from glucose oxidation under normoxic conditions in GCN5L1-depleted cells. We show that GCN5L1 directly binds to the Akt-activating mTORC2 component Rictor, and that loss of Rictor acetylation is evident in GCN5L1 knockdown cells. Finally, we show that restoring Rictor acetylation in GCN5L1-depleted cells reduces mitochondrial ROS generation and increases cell survival in response to H/R. These studies suggest that GCN5L1 may play a central role in energy substrate metabolism and cell survival via the regulation of Akt/mTORC2 signaling.

Cardiac-specific deletion of GCN5L1 restricts recovery from ischemia-reperfusion injury.

GCN5L1 regulates mitochondrial protein acetylation, cellular bioenergetics, reactive oxygen species (ROS) generation, and organelle positioning in a number of diverse cell types. However, the functional role of GCN5L1 in the heart is currently unknown. As many of the factors regulated by GCN5L1 play a major role in ischemia-reperfusion (I/R) injury, we sought to determine if GCN5L1 is an important nexus in the response to cardiac ischemic stress. Deletion of GCN5L1 in cardiomyocytes resulted in impaired myocardial post-ischemic function and increased infarct development in isolated work-performing hearts. GCN5L1 knockout hearts displayed hallmarks of ROS damage, and scavenging of ROS restored cardiac function and reduced infarct volume in vivo. GCN5L1 knockdown in cardiac-derived AC16 cells was associated with reduced activation of the pro-survival MAP kinase ERK1/2, which was also reversed by ROS scavenging, leading to restored cell viability. We therefore conclude that GCN5L1 activity provides an important protection against I/R induced, ROS-mediated damage in the ischemic heart.

Adropin treatment restores cardiac glucose oxidation in pre-diabetic obese mice.

Exposure to a high fat (HF) diet promotes increased fatty acid uptake, fatty acid oxidation and lipid accumulation in the heart. These maladaptive changes impact cellular energy metabolism and may promote the development of cardiac dysfunction. Attempts to increase cardiac glucose utilization have been proposed as a way to reverse cardiomyopathy in obese and diabetic individuals. Adropin is a nutrient-regulated metabolic hormone shown to promote glucose oxidation over fatty acid oxidation in skeletal muscle homogenates in vitro. The focus of the current study was to investigate whether adropin can regulate substrate metabolism in the heart following prolonged exposure to a HF diet in vivo. Mice on a long-term HF diet received serial intraperitoneal injections of vehicle or adropin over three days. Cardiac glucose oxidation was significantly reduced in HF animals, which was rescued by acute adropin treatment. Significant decreases in cardiac pyruvate dehydrogenase activity were observed in HF animals, which were also reversed by adropin treatment. In contrast to previous studies, this change was unrelated to Pdk4 expression, which remained elevated in both vehicle- and adropin-treated HF mice. Instead, we show that adropin modulated the expression of the mitochondrial acetyltransferase enzyme GCN5L1, which altered the acetylation status and activity of fuel metabolism enzymes to favor glucose utilization. Our findings indicate that adropin exposure leads to increased cardiac glucose oxidation under HF conditions, and may provide a future therapeutic avenue in the treatment of diabetic cardiomyopathy.

The protein acetylase GCN5L1 modulates hepatic fatty acid oxidation activity via acetylation of the mitochondrial ß-oxidation enzyme HADHA.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1.NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice.

Loss of Rad-GTPase produces a novel adaptive cardiac phenotype resistant to systolic decline with aging.

Rad-GTPase is a regulator of L-type calcium current (LTCC), with increased calcium current observed in Rad knockout models. While mouse models that result in elevated LTCC have been associated with heart failure, our laboratory and others observe a hypercontractile phenotype with enhanced calcium homeostasis in Rad(-/-). It is currently unclear whether this observation represents an early time point in a decompensatory progression towards heart failure or whether Rad loss drives a novel phenotype with stable enhanced function. We test the hypothesis that Rad(-/-) drives a stable nonfailing hypercontractile phenotype in adult hearts, and we examine compensatory regulation of sarcoplasmic reticulum (SR) loading and protein changes. Heart function was measured in vivo with echocardiography. In vivo heart function was significantly improved in adult Rad(-/-) hearts compared with wild type. Heart wall dimensions were significantly increased, while heart size was decreased, and cardiac output was not changed. Cardiac function was maintained through 18 mo of age with no decompensation. SR releasable Ca(2+) was increased in isolated Rad(-/-) ventricular myocytes. Higher Ca(2+) load was accompanied by sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) protein elevation as determined by immunoblotting and a rightward shift in the thapsigargan inhibitor-response curve. Rad(-/-) promotes morphological changes accompanied by a stable increase in contractility with aging and preserved cardiac output. The Rad(-/-) phenotype is marked by enhanced systolic and diastolic function with increased SR uptake, which is consistent with a model that does not progress into heart failure.

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress.

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

Quantitative phosphoproteomics using acetone-based peptide labeling: method evaluation and application to a cardiac ischemia/reperfusion model.

Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. An MS-based strategy utilizing an inexpensive acetone-based peptide-labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Because the chemistry for RABA labeling for phosphorylation profiling had not been previously reported, it was first validated using a standard phosphoprotein and identical phosphoproteomes from cardiac tissue extracts. A workflow was then utilized to compare cardiac tissue phosphoproteomes from mouse hearts not expressing FGF2 versus hearts expressing low-molecular-weight fibroblast growth factor-2 (LMW FGF2) to relate low-molecular-weight fibroblast growth factor-2 (LMW FGF2)-mediated cardioprotective phenomena induced by ischemia/reperfusion injury of hearts, with downstream phosphorylation changes in LMW FGF2 signaling cascades. Statistically significant phosphorylation changes were identified at 14 different sites on 10 distinct proteins, including some with mechanisms already established for LMW FGF2-mediated cardioprotective signaling (e.g., connexin-43), some with new details linking LMW FGF2 to the cardioprotective mechanisms (e.g., cardiac myosin binding protein C or cMyBPC), and also several new downstream effectors not previously recognized for cardio-protective signaling by LMW FGF2. Additionally, one of the phosphopeptides, cMyBPC/pSer-282, identified was further verified with site-specific quantification using an SRM (selected reaction monitoring)-based approach that also relies on isotope labeling of a synthetic phosphopeptide with deuterated acetone as an internal standard. Overall, this study confirms that the inexpensive acetone-based peptide labeling can be used in both exploratory and targeted quantification phosphoproteomic studies to identify and verify biologically relevant phosphorylation changes in whole tissues.

Low molecular weight fibroblast growth factor-2 signals via protein kinase C and myofibrillar proteins to protect against postischemic cardiac dysfunction.

Among its many biological roles, fibroblast growth factor-2 (FGF2) acutely protects the heart from dysfunction associated with ischemia/reperfusion (I/R) injury. Our laboratory has demonstrated that this is due to the activity of the low molecular weight (LMW) isoform of FGF2 and that FGF2-mediated cardioprotection relies on the activity of protein kinase C (PKC); however, which PKC isoforms are responsible for LMW FGF2-mediated cardioprotection, and their downstream targets, remain to be elucidated. To identify the PKC pathway(s) that contributes to postischemic cardiac recovery by LMW FGF2, mouse hearts expressing only LMW FGF2 (HMWKO) were bred to mouse hearts not expressing PKCα (PKCαKO) or subjected to a selective PKCε inhibitor (εV(1-2)) before and during I/R. Hearts only expressing LMW FGF2 showed significantly improved postischemic recovery of cardiac function following I/R (P < 0.05), which was significantly abrogated in the absence of PKCα (P < 0.05) or presence of PKCε inhibition (P < 0.05). Hearts only expressing LMW FGF2 demonstrated differences in actomyosin ATPase activity as well as increases in the phosphorylation of troponin I and T during I/R compared with wild-type hearts; several of these effects were dependent on PKCα activity. This evidence indicates that both PKCα and PKCε play a role in LMW FGF2-mediated protection from cardiac dysfunction and that PKCα signaling to the contractile apparatus is a key step in the mechanism of LMW FGF2-mediated protection against myocardial dysfunction.

Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue.

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in a number of key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress.

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.

General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

G-protein coupled receptor 19 (GPR19) knockout mice display sex-dependent metabolic dysfunction.

G-protein coupled receptors (GPCRs) mediate signal transduction from the cellular surface to intracellular metabolic pathways. While the function of many GPCRs has been delineated previously, a significant number require further characterization to elucidate their cellular function. G-protein coupled receptor 19 (GPR19) is a poorly characterized class A GPCR which has been implicated in the regulation of circadian rhythm, tumor metastasis, and mitochondrial homeostasis. In this report, we use a novel knockout (KO) mouse model to examine the role of GPR19 in whole-body metabolic regulation. We show that loss of GPR19 promotes increased energy expenditure and decreased activity in both male and female mice. However, only male GPR19 KO mice display glucose intolerance in response to a high fat diet. Loss of GPR19 expression in male mice, but not female mice, resulted in diet-induced hepatomegaly, which was associated with decreased expression of key fatty acid oxidation genes in male GPR19 KO livers. Overall, our data suggest that loss of GPR19 impacts whole-body energy metabolism in diet-induced obese mice in a sex-dependent manner.

Myocardial brain-derived neurotrophic factor regulates cardiac bioenergetics through the transcription factor Yin Yang 1.

AIMS: Brain-derived neurotrophic factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, tropomyosin-related kinase receptor (TrkB), are expressed in cardiomyocytes; however, the role of myocardial BDNF signalling in cardiac pathophysiology is poorly understood. Here, we investigated the role of BDNF/TrkB signalling in cardiac stress response to exercise and pathological stress. METHODS AND RESULTS: We found that myocardial BDNF expression was increased in mice with swimming exercise but decreased in a mouse heart failure model and human failing hearts. Cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). In response to pathological stress (transaortic constriction, TAC), cTrkB KO mice showed an exacerbated heart failure progression. The downregulation of PGC-1α in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unravelled that BDNF induces PGC-1α upregulation and bioenergetics through a novel signalling pathway, the pleiotropic transcription factor Yin Yang 1. CONCLUSION: Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

Fibroblast growth factor-2-induced cardioprotection against myocardial infarction occurs via the interplay between nitric oxide, protein kinase signaling, and ATP-sensitive potassium channels.

Fibroblast growth factor-2 (FGF2) protects the heart from ischemia-reperfusion (I-R) injury via a vast network of protein kinases. In the heart, downstream effectors of these FGF2-triggered signals have not yet been identified. It is hypothesized that nitric oxide (NO) signaling and ATP-sensitive potassium (K(ATP)) channel activity are key effectors of protein kinases activated by FGF2-mediated cardioprotection. Hearts with a cardiac-specific overexpression of FGF2 (FGF2 Tg) were subjected to I-R injury in the absence or the presence of selective inhibitors of NO synthase (NOS) isoforms or sarcolemmal (sarcK(ATP)) and mitochondrial (mitoK(ATP)) K(ATP) channels. Multiple NOS isoforms are necessary for FGF2-mediated cardioprotection, and nitrite levels are significantly reduced in FGF2 Tg hearts upon inhibition of protein kinase C or mitogen-activated protein kinases. Likewise, sarcK(ATP) and mitoK(ATP) channels are important for cardioprotection elicited by endogenous FGF2. These findings suggest that FGF2-induced cardioprotection occurs via protein kinase-NOS pathways as well as K(ATP) channel activity.

Diet-induced obese mice are resistant to improvements in cardiac function resulting from short-term adropin treatment.

Previous studies have shown that treatment with recombinant adropin, a circulating peptide secreted by the liver and brain, restores glucose utilization in the hearts of diet-induced obese mice. This restoration of fuel substrate flexibility, which is lost in obese and diabetic animals, has the potential to improve contractile function in the diabetic heart. Using an ex vivo approach, we examined whether short-term adropin treatment could enhance cardiac function in a mouse model of diet-induced obesity. Our study showed that acute adropin treatment reduces inhibitory phosphorylation of pyruvate dehydrogenase in primary neonatal cardiomyocytes, and leads to moderate improvements in ex vivo cardiac function in mice fed a low fat diet. Conversely, short-term exposure to adropin led to a small decrease in cardiac function in mice fed a long-term high fat diet. Insulin treatment did not significantly alter cardiac function in adropin treated hearts from either low or high fat diet mice, however acute adropin treatment did moderately restore some aspects of downstream insulin signaling in high fat diet fed mice. Overall, these data suggest that in an ex vivo setting, acute adropin treatment alone is not sufficient to promote improved cardiac function in obese animals.